Single cell spatial analysis reveals inflammatory foci of immature neutrophil and CD8 T cells in COVID-19 lungs.

Single cell spatial interrogation of the immune-structural interactions in COVID -19 lungs is challenging, mainly because of the marked cellular infiltrate and architecturally distorted microstructure. To address this, we develop a suite of mathematical tools to search for statistically significant co-locations amongst immune and structural cells identified using 37-plex imaging mass cytometry. This unbiased method reveals a cellular map interleaved with an inflammatory network of immature neutrophils, cytotoxic CD8 T cells, megakaryocytes and monocytes co-located with regenerating alveolar progenitors and endothelium. Of note, a highly active cluster of immature neutrophils and CD8 T cells, is found spatially linked with alveolar progenitor cells, and temporally with the diffuse alveolar damage stage. These findings offer further insights into how immune cells interact in the lungs of severe COVID-19 disease. We provide our pipeline [Spatial Omics Oxford Pipeline (SpOOx)] and visual-analytical tool, Multi-Dimensional Viewer (MDV) software, as a resource for spatial analysis.

Breathlessness in a virtual world: An experimental paradigm testing how discrepancy between VR visual gradients and pedal resistance during stationary cycling affects breathlessness perception.

INTRODUCTION: The sensation of breathlessness is often attributed to perturbations in cardio-pulmonary physiology, leading to changes in afferent signals. New evidence suggests that these signals are interpreted in the light of prior "expectations". A misalignment between afferent signals and expectations may underly unexplained breathlessness. Using a novel immersive virtual reality (VR) exercise paradigm, we investigated whether manipulating an individual's expectation of effort (determined by a virtual hill gradient) may alter their perception of breathlessness, independent from actual effort (the physical effort of cycling). METHODS: Nineteen healthy volunteers completed a single experimental session where they exercised on a cycle ergometer while wearing a VR headset. We created an immersive virtual cycle ride where participants climbed up 100 m hills with virtual gradients of 4%, 6%, 8%, 10% and 12%. Each virtual hill gradient was completed twice: once with a 4% cycling ergometer resistance and once with a 6% resistance, allowing us to dissociate expected effort (virtual hill gradient) from actual effort (power). At the end of each hill, participants reported their perceived breathlessness. Linear mixed effects models were used to examine the independent contribution of actual effort and expected effort to ratings of breathlessness (0-10 scale). RESULTS: Expectation of effort (effect estimate ± std. error, 0.63 ± 0.11, P < 0.001) and actual effort (0.81 ± 0.21, P < 0.001) independently explained subjective ratings of breathlessness, with comparable contributions of 19% and 18%, respectively. Additionally, we found that effort expectation accounted for 6% of participants' power and was a significant, independent predictor (0.09 ± 0.03; P = 0.001). CONCLUSIONS: An individuals' expectation of effort is equally important for forming perceptions of breathlessness as the actual effort required to cycle. A new VR paradigm enables this to be experimentally studied and could be used to re-align breathlessness and enhance training programmes.

LanceOtron: a deep learning peak caller for genome sequencing experiments.

MOTIVATION: Genome sequencing experiments have revolutionized molecular biology by allowing researchers to identify important DNA-encoded elements genome wide. Regions where these elements are found appear as peaks in the analog signal of an assay's coverage track, and despite the ease with which humans can visually categorize these patterns, the size of many genomes necessitates algorithmic implementations. Commonly used methods focus on statistical tests to classify peaks, discounting that the background signal does not completely follow any known probability distribution and reducing the information-dense peak shapes to simply maximum height. Deep learning has been shown to be highly accurate for many pattern recognition tasks, on par or even exceeding human capabilities, providing an opportunity to reimagine and improve peak calling. RESULTS: We present the peak calling framework LanceOtron, which combines deep learning for recognizing peak shape with multifaceted enrichment calculations for assessing significance. In benchmarking ATAC-seq, ChIP-seq and DNase-seq, LanceOtron outperforms long-standing, gold-standard peak callers through its improved selectivity and near-perfect sensitivity. AVAILABILITY AND IMPLEMENTATION: A fully featured web application is freely available from LanceOtron.molbiol.ox.ac.uk, command line interface via python is pip installable from PyPI at https://pypi.org/project/lanceotron/, and source code and benchmarking tests are available at https://github.com/LHentges/LanceOtron. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Multi Locus View: an extensible web-based tool for the analysis of genomic data.

Tracking and understanding data quality, analysis and reproducibility are critical concerns in the biological sciences. This is especially true in genomics where next generation sequencing (NGS) based technologies such as ChIP-seq, RNA-seq and ATAC-seq are generating a flood of genome-scale data. However, such data are usually processed with automated tools and pipelines, generating tabular outputs and static visualisations. Interpretation is normally made at a high level without the ability to visualise the underlying data in detail. Conventional genome browsers are limited to browsing single locations and do not allow for interactions with the dataset as a whole. Multi Locus View (MLV), a web-based tool, has been developed to allow users to fluidly interact with genomics datasets at multiple scales. The user is able to browse the raw data, cluster, and combine the data with other analysis and annotate the data. User datasets can then be shared with other users or made public for quick assessment from the academic community. MLV is publically available at https://mlv.molbiol.ox.ac.uk .

High-resolution targeted 3C interrogation of cis-regulatory element organization at genome-wide scale.

Chromosome conformation capture (3C) provides an adaptable tool for studying diverse biological questions. Current 3C methods generally provide either low-resolution interaction profiles across the entire genome, or high-resolution interaction profiles at limited numbers of loci. Due to technical limitations, generation of reproducible high-resolution interaction profiles has not been achieved at genome-wide scale. Here, to overcome this barrier, we systematically test each step of 3C and report two improvements over current methods. We show that up to 30% of reporter events generated using the popular in situ 3C method arise from ligations between two individual nuclei, but this noise can be almost entirely eliminated by isolating intact nuclei after ligation. Using Nuclear-Titrated Capture-C, we generate reproducible high-resolution genome-wide 3C interaction profiles by targeting 8055 gene promoters in erythroid cells. By pairing high-resolution 3C interaction calls with nascent gene expression we interrogate the role of promoter hubs and super-enhancers in gene regulation.

Genomic approaches used to investigate an atypical outbreak of Salmonella Adjame.

In 2017, an outbreak of gastroenteritis in England attributed to Salmonella Adjame was detected and investigated. With the introduction of whole genome sequencing (WGS) for microbial typing, methods for comparing international outbreak data require evaluation. A case was defined as a person resident in England with a clinical sample from 1 June 2017 to 27 July 2017 from whom S. Adjame was isolated. Cases were interviewed and exposures analysed. Backward tracing of food provenance was undertaken. WGS was performed on isolates from cases and historical isolates and compared using Public Health England's SnapperDB high-quality SNP pipeline and Enterobase's Salmonella core genome multi-locus sequence typing (cgMLST) scheme. In total, 14 cases were identified. The majority were vegetarian, probably of South Asian descent, with a median age of 66.5 years with no recent international travel reported. Cases consumed a range of fresh food products including herbs and spices bought from South Asian grocers. Backward tracing did not identify a common source. WGS typing showed sub-clustering and considerable genetic variation across human samples. cgMLST allele-based analysis was comparable to SNP-derived phylogenetic analysis and clusters were defined using each method. Imported herbs or spices were suspected vehicles. The cases were linked in time and place but WGS showed marked heterogeneity, atypical of a point source Salmonella outbreak. The application of incorporating SNP or allelic differences into the case definition may not always be appropriate. With further validation, cgMLST could be used for international outbreak alerts when WGS analysis is being undertaken to facilitate comparison.

GrapeTree: visualization of core genomic relationships among 100,000 bacterial pathogens.

Current methods struggle to reconstruct and visualize the genomic relationships of large numbers of bacterial genomes. GrapeTree facilitates the analyses of large numbers of allelic profiles by a static "GrapeTree Layout" algorithm that supports interactive visualizations of large trees within a web browser window. GrapeTree also implements a novel minimum spanning tree algorithm (MSTree V2) to reconstruct genetic relationships despite high levels of missing data. GrapeTree is a stand-alone package for investigating phylogenetic trees plus associated metadata and is also integrated into EnteroBase to facilitate cutting edge navigation of genomic relationships among bacterial pathogens.

Pan-genome Analysis of Ancient and Modern Salmonella enterica Demonstrates Genomic Stability of the Invasive Para C Lineage for Millennia.

Salmonella enterica serovar Paratyphi C causes enteric (paratyphoid) fever in humans. Its presentation can range from asymptomatic infections of the blood stream to gastrointestinal or urinary tract infection or even a fatal septicemia [1]. Paratyphi C is very rare in Europe and North America except for occasional travelers from South and East Asia or Africa, where the disease is more common [2, 3]. However, early 20th-century observations in Eastern Europe [3, 4] suggest that Paratyphi C enteric fever may once have had a wide-ranging impact on human societies. Here, we describe a draft Paratyphi C genome (Ragna) recovered from the 800-year-old skeleton (SK152) of a young woman in Trondheim, Norway. Paratyphi C sequences were recovered from her teeth and bones, suggesting that she died of enteric fever and demonstrating that these bacteria have long caused invasive salmonellosis in Europeans. Comparative analyses against modern Salmonella genome sequences revealed that Paratyphi C is a clade within the Para C lineage, which also includes serovars Choleraesuis, Typhisuis, and Lomita. Although Paratyphi C only infects humans, Choleraesuis causes septicemia in pigs and boar [5] (and occasionally humans), and Typhisuis causes epidemic swine salmonellosis (chronic paratyphoid) in domestic pigs [2, 3]. These different host specificities likely evolved in Europe over the last ∼4,000 years since the time of their most recent common ancestor (tMRCA) and are possibly associated with the differential acquisitions of two genomic islands, SPI-6 and SPI-7. The tMRCAs of these bacterial clades coincide with the timing of pig domestication in Europe [6].

A genomic overview of the population structure of Salmonella.

For many decades, Salmonella enterica has been subdivided by serological properties into serovars or further subdivided for epidemiological tracing by a variety of diagnostic tests with higher resolution. Recently, it has been proposed that so-called eBurst groups (eBGs) based on the alleles of seven housekeeping genes (legacy multilocus sequence typing [MLST]) corresponded to natural populations and could replace serotyping. However, this approach lacks the resolution needed for epidemiological tracing and the existence of natural populations had not been independently validated by independent criteria. Here, we describe EnteroBase, a web-based platform that assembles draft genomes from Illumina short reads in the public domain or that are uploaded by users. EnteroBase implements legacy MLST as well as ribosomal gene MLST (rMLST), core genome MLST (cgMLST), and whole genome MLST (wgMLST) and currently contains over 100,000 assembled genomes from Salmonella. It also provides graphical tools for visual interrogation of these genotypes and those based on core single nucleotide polymorphisms (SNPs). eBGs based on legacy MLST are largely consistent with eBGs based on rMLST, thus demonstrating that these correspond to natural populations. rMLST also facilitated the selection of representative genotypes for SNP analyses of the entire breadth of diversity within Salmonella. In contrast, cgMLST provides the resolution needed for epidemiological investigations. These observations show that genomic genotyping, with the assistance of EnteroBase, can be applied at all levels of diversity within the Salmonella genus.

Impact of human-associated Escherichia coli clonal groups in Antarctic pinnipeds: presence of ST73, ST95, ST141 and ST131.

There is growing concern about the spreading of human microorganisms in relatively untouched ecosystems such as the Antarctic region. For this reason, three pinniped species (Leptonychotes weddellii, Mirounga leonina and Arctocephalus gazella) from the west coast of the Antartic Peninsula were analysed for the presence of Escherichia spp. with the recovery of 158 E. coli and three E. albertii isolates. From those, 23 harboured different eae variants (α1, ß1, ß2, ε1, θ1, κ, ο), including a bfpA-positive isolate (O49:H10-A-ST206, eae-k) classified as typical enteropathogenic E. coli. Noteworthy, 62 of the 158 E. coli isolates (39.2%) exhibited the ExPEC status and 27 (17.1%) belonged to sequence types (ST) frequently occurring among urinary/bacteremia ExPEC clones: ST12, ST73, ST95, ST131 and ST141. We found similarities >85% within the PFGE-macrorrestriction profiles of pinniped and human clinic O2:H6-B2-ST141 and O16:H5/O25b:H4-B2-ST131 isolates. The in silico analysis of ST131 Cplx genomes from the three pinnipeds (five O25:H4-ST131/PST43-fimH22-virotype D; one O16:H5-ST131/PST506-fimH41; one O25:H4-ST6252/PST9-fimH22-virotype D1) identified IncF and IncI1 plasmids and revealed high core-genome similarities between pinniped and human isolates (H22 and H41 subclones). This is the first study to demonstrate the worrisome presence of human-associated E. coli clonal groups, including ST131, in Antarctic pinnipeds.

Promotion of Germination Using Hydroxamic Acid Inhibitors of 9-cis-Epoxycarotenoid Dioxygenase.

Abscisic acid (ABA) inhibits seed germination and the regulation of ABA biosynthesis has a role in maintenance of seed dormancy. The key rate-limiting step in ABA biosynthesis is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). Two hydroxamic acid inhibitors of carotenoid cleavage dioxygenase (CCD), D4 and D7, previously found to inhibit CCD and NCED in vitro, are shown to have the novel property of decreasing mean germination time of tomato (Solanum lycopersicum L.) seeds constitutively overexpressing LeNCED1. Post-germination, D4 exhibited no negative effects on tomato seedling growth in terms of height, dry weight, and fresh weight. Tobacco (Nicotiana tabacum L.) seeds containing a tetracycline-inducible LeNCED1 transgene were used to show that germination could be negatively and positively controlled through the chemical induction of gene expression and the chemical inhibition of the NCED protein: application of tetracycline increased mean germination time and delayed hypocotyl emergence in a similar manner to that observed when exogenous ABA was applied and this was reversed by D4 when NCED expression was induced at intermediate levels. D4 also improved germination in lettuce (Lactuca sativa L.) seeds under thermoinhibitory temperatures and in tomato seeds imbibed in high osmolarity solutions of polyethylene glycol. D4 reduced ABA and dihydrophaseic acid accumulation in tomato seeds overexpressing LeNCED1 and reduced ABA accumulation in wild type tomato seeds imbibed on polyethylene glycol. The evidence supports a mode of action of D4 through NCED inhibition, and this molecule provides a lead compound for the design of NCED inhibitors with greater specificity and potency.

Draft Genome Sequences of Six Novel Bacterial Isolates from Chicken Ceca.

The chicken is the most common domesticated animal and the most abundant bird in the world. However, the chicken gut is home to many previously uncharacterized bacterial taxa. Here, we report draft genome sequences from six bacterial isolates from chicken ceca, all of which fall outside any named species.

Differences in the Faecal Microbiome in Schistosoma haematobium Infected Children vs. Uninfected Children.

BACKGROUND: Several infectious diseases and therapeutic interventions cause gut microbe dysbiosis and associated pathology. We characterised the gut microbiome of children exposed to the helminth Schistosoma haematobium pre- and post-treatment with the drug praziquantel (PZQ), with the aim to compare the gut microbiome structure (abundance and diversity) in schistosome infected vs. uninfected children. METHODS: Stool DNA from 139 children aged six months to 13 years old; with S. haematobium infection prevalence of 27.34% was extracted at baseline. 12 weeks following antihelminthic treatment with praziqunatel, stool DNA was collected from 62 of the 139 children. The 16S rRNA genes were sequenced from the baseline and post-treatment samples and the sequence data, clustered into operational taxonomic units (OTUs). The OTU data were analysed using multivariate analyses and paired T-test. RESULTS: Pre-treatment, the most abundant phyla were Bacteroidetes, followed by Firmicutes and Proteobacteria respectively. The relative abundance of taxa among bacterial classes showed limited variation by age group or sex and the bacterial communities had similar overall compositions. Although there were no overall differences in the microbiome structure across the whole age range, the abundance of 21 OTUs varied significantly with age (FDR<0.05). Some OTUs including Veillonella, Streptococcus, Bacteroides and Helicobacter were more abundant in children ≤ 1 year old compared to older children. Furthermore, the gut microbiome differed in schistosome infected vs. uninfected children with 27 OTU occurring in infected but not uninfected children, for 5 of these all Prevotella, the difference was statistically significant (p <0.05) with FDR <0.05. PZQ treatment did not alter the microbiome structure in infected or uninfected children from that observed at baseline. CONCLUSIONS: There are significant differences in the gut microbiome structure of infected vs. uninfected children and the differences were refractory to PZQ treatment.

Eighteenth-century genomes show that mixed infections were common at time of peak tuberculosis in Europe.

Tuberculosis (TB) was once a major killer in Europe, but it is unclear how the strains and patterns of infection at 'peak TB' relate to what we see today. Here we describe 14 genome sequences of M. tuberculosis, representing 12 distinct genotypes, obtained from human remains from eighteenth-century Hungary using metagenomics. All our historic genotypes belong to M. tuberculosis Lineage 4. Bayesian phylogenetic dating, based on samples with well-documented dates, places the most recent common ancestor of this lineage in the late Roman period. We find that most bodies yielded more than one M. tuberculosis genotype and we document an intimate epidemiological link between infections in two long-dead individuals. Our results suggest that metagenomic approaches usefully inform detection and characterization of historical and contemporary infections.

Resequencing at ≥40-Fold Depth of the Parental Genomes of a Solanum lycopersicum × S. pimpinellifolium Recombinant Inbred Line Population and Characterization of Frame-Shift InDels That Are Highly Likely to Perturb Protein Function.

A recombinant in-bred line population derived from a cross between Solanum lycopersicum var. cerasiforme (E9) and S. pimpinellifolium (L5) has been used extensively to discover quantitative trait loci (QTL), including those that act via rootstock genotype, however, high-resolution single-nucleotide polymorphism genotyping data for this population are not yet publically available. Next-generation resequencing of parental lines allows the vast majority of polymorphisms to be characterized and used to progress from QTL to causative gene. We sequenced E9 and L5 genomes to 40- and 44-fold depth, respectively, and reads were mapped to the reference Heinz 1706 genome. In L5 there were three clear regions on chromosome 1, chromosome 4, and chromosome 8 with increased rates of polymorphism. Two other regions were highly polymorphic when we compared Heinz 1706 with both E9 and L5 on chromosome 1 and chromosome 10, suggesting that the reference sequence contains a divergent introgression in these locations. We also identified a region on chromosome 4 consistent with an introgression from S. pimpinellifolium into Heinz 1706. A large dataset of polymorphisms for the use in fine-mapping QTL in a specific tomato recombinant in-bred line population was created, including a high density of InDels validated as simple size-based polymerase chain reaction markers. By careful filtering and interpreting the SnpEff prediction tool, we have created a list of genes that are predicted to have highly perturbed protein functions in the E9 and L5 parental lines.

Culture-independent detection and characterisation of Mycobacterium tuberculosis and M. africanum in sputum samples using shotgun metagenomics on a benchtop sequencer.

Tuberculosis remains a major global health problem. Laboratory diagnostic methods that allow effective, early detection of cases are central to management of tuberculosis in the individual patient and in the community. Since the 1880s, laboratory diagnosis of tuberculosis has relied primarily on microscopy and culture. However, microscopy fails to provide species- or lineage-level identification and culture-based workflows for diagnosis of tuberculosis remain complex, expensive, slow, technically demanding and poorly able to handle mixed infections. We therefore explored the potential of shotgun metagenomics, sequencing of DNA from samples without culture or target-specific amplification or capture, to detect and characterise strains from the Mycobacterium tuberculosis complex in smear-positive sputum samples obtained from The Gambia in West Africa. Eight smear- and culture-positive sputum samples were investigated using a differential-lysis protocol followed by a kit-based DNA extraction method, with sequencing performed on a benchtop sequencing instrument, the Illumina MiSeq. The number of sequence reads in each sputum-derived metagenome ranged from 989,442 to 2,818,238. The proportion of reads in each metagenome mapping against the human genome ranged from 20% to 99%. We were able to detect sequences from the M. tuberculosis complex in all eight samples, with coverage of the H37Rv reference genome ranging from 0.002X to 0.7X. By analysing the distribution of large sequence polymorphisms (deletions and the locations of the insertion element IS6110) and single nucleotide polymorphisms (SNPs), we were able to assign seven of eight metagenome-derived genomes to a species and lineage within the M. tuberculosis complex. Two metagenome-derived mycobacterial genomes were assigned to M. africanum, a species largely confined to West Africa; the others that could be assigned belonged to lineages T, H or LAM within the clade of "modern" M. tuberculosis strains. We have provided proof of principle that shotgun metagenomics can be used to detect and characterise M. tuberculosis sequences from sputum samples without culture or target-specific amplification or capture, using an accessible benchtop-sequencing platform, the Illumina MiSeq, and relatively simple DNA extraction, sequencing and bioinformatics protocols. In our hands, sputum metagenomics does not yet deliver sufficient depth of coverage to allow sequence-based sensitivity testing; it remains to be determined whether improvements in DNA extraction protocols alone can deliver this or whether culture, capture or amplification steps will be required. Nonetheless, we can foresee a tipping point when a unified automated metagenomics-based workflow might start to compete with the plethora of methods currently in use in the diagnostic microbiology laboratory.

Recovery of a medieval Brucella melitensis genome using shotgun metagenomics.

Shotgun metagenomics provides a powerful assumption-free approach to the recovery of pathogen genomes from contemporary and historical material. We sequenced the metagenome of a calcified nodule from the skeleton of a 14th-century middle-aged male excavated from the medieval Sardinian settlement of Geridu. We obtained 6.5-fold coverage of a Brucella melitensis genome. Sequence reads from this genome showed signatures typical of ancient or aged DNA. Despite the relatively low coverage, we were able to use information from single-nucleotide polymorphisms to place the medieval pathogen genome within a clade of B. melitensis strains that included the well-studied Ether strain and two other recent Italian isolates. We confirmed this placement using information from deletions and IS711 insertions. We conclude that metagenomics stands ready to document past and present infections, shedding light on the emergence, evolution, and spread of microbial pathogens. Importance: Infectious diseases have shaped human populations and societies throughout history. The recovery of pathogen DNA sequences from human remains provides an opportunity to identify and characterize the causes of individual and epidemic infections. By sequencing DNA extracted from medieval human remains through shotgun metagenomics, without target-specific capture or amplification, we have obtained a draft genome sequence of an ~700-year-old Brucella melitensis strain. Using a variety of bioinformatic approaches, we have shown that this historical strain is most closely related to recent strains isolated from Italy, confirming the continuity of this zoonotic infection, and even a specific lineage, in the Mediterranean region over the centuries.

Extensive microbial and functional diversity within the chicken cecal microbiome.

Chickens are major source of food and protein worldwide. Feed conversion and the health of chickens relies on the largely unexplored complex microbial community that inhabits the chicken gut, including the ceca. We have carried out deep microbial community profiling of the microbiota in twenty cecal samples via 16S rRNA gene sequences and an in-depth metagenomics analysis of a single cecal microbiota. We recovered 699 phylotypes, over half of which appear to represent previously unknown species. We obtained 648,251 environmental gene tags (EGTs), the majority of which represent new species. These were binned into over two-dozen draft genomes, which included Campylobacter jejuni and Helicobacter pullorum. We found numerous polysaccharide- and oligosaccharide-degrading enzymes encoding within the metagenome, some of which appeared to be part of polysaccharide utilization systems with genetic evidence for the co-ordination of polysaccharide degradation with sugar transport and utilization. The cecal metagenome encodes several fermentation pathways leading to the production of short-chain fatty acids, including some with novel features. We found a dozen uptake hydrogenases encoded in the metagenome and speculate that these provide major hydrogen sinks within this microbial community and might explain the high abundance of several genera within this microbiome, including Campylobacter, Helicobacter and Megamonas.